This invention relates generally to nucleic acid hybridization analysis. More specifically, an oligonucleotide probe for hybridization analysis is provided, that comprises a nucleotide sequence which, under suitable conditions, is capable of forming a hairpin structure. The double stranded segment of the hairpin structure is formed between two perfectly matched nucleotide sequences, and at least a portion of the nucleotide sequence within the double stranded segment is complementary to a target nucleotide sequence to be detected. An array of oligonucleotide probes immobilized on a solid support for hybridization analysis is also provided that comprises a solid support suitable for use in nucleic acid hybridization having immobilized thereon a plurality of oligonucleotide probes. The probes each comprise a nucleotide sequence which, under suitable conditions, is capable of forming a hairpin structure having a nucleotide sequence complementary to a target nucleotide sequence to be detected located within the double stranded segment. Methods for nucleic acid hybridization analysis using the probes or array of immobilized probes are additionally provided.
Nucleic acid hybridization, in the forty years since its discovery, has become a powerful tool with implications for biology, medicine and industry. Hybridization assays are based on the very specific base pairing that is found in hybrids of DNA and RNA. Base sequences of analytical interest appearing along a strand of nucleic acid can be detected very specifically and sensitively by observing the formation of hybrids in the presence of a probe nucleic acid known to comprise a base sequence that is complementary with the sequence of interest. Nucleic acid hybridization has been used for a wide variety of purposes including, for example, identification of specific clones from cDNA and genomic libraries, detecting single base pair polymorphisms in DNA, generating mutations by oligonucleotide mutagenesis, amplifying nucleic acids from single cells or viruses, or detecting microbial infections.
Recent advances in nucleic acid hybridization methods have greatly expanded the scope and extent of its potential applications. Of great interest are approaches to miniaturize hybridization reactions by preparing xe2x80x9cmicroarray biochipsxe2x80x9d (or xe2x80x9cDNA chipsxe2x80x9d) containing large numbers of oligonucleotide probes prepared, for example, through VLSIPSTM technology (See U.S. Pat. Nos. 5,143,854 or 5,561,071). These approaches offer great promise for a wide variety of applications. Microarray biochips are useful for sequencing nucleic acid by hybridization (see, for example, U.S. Pat. No. 5,741,644), for diagnosis of human immunodeficiency virus (see, for example, U.S. Pat. No. 5,861,242) and for screening potential DNA binding drugs (see, for example, U.S. Pat. No. 5,556,752).
When using nucleic acid microarrays, there are two general approaches for detecting hybridization to a nucleic acid. Detection can be accomplished if the target nucleic acid is labeled (xe2x80x9cdirect labeling approachxe2x80x9d). Alternatively, detection can be accomplished by a second probe that is detectably labeled and which can hybridize to the nucleic acid of the sample, which is hybridized to the first probe immobilized on the array (xe2x80x9cindirectxe2x80x9d labeling approach).
Bagwell, U.S. Pat. No. 5,607,834 discloses a fluorescent probe for binding to a polynucleotide target and methods using such fluorescent probes that comprises: an oligonucleotide having a segment complementary to the polynucleotide target, the oligonucleotide forming two imperfect hairpins both of which together include the segment except for one nucleotide; and one donor fluorophore and one acceptor fluorophore covalently attached to the oligonucleotide so that only when the imperfect hairpins are formed, the donor fluorophore and the acceptor fluorophore are in close proximity to allow resonance energy transfer therebetween. The fluorescent probes disclosed in Bagwell must contain xe2x80x9cimperfect hairpins,xe2x80x9d i.e., containing mismatches in the double-stranded stem segment. In addition, Bagwell does not disclose or teach any immobilized arrays of oligonucleotide probes.
Nazarenko et al., U.S. Pat. No. 5,866,336 disclose an oligonucleotide containing a hairpin structure for use as a primer in detecting a target nucleotide sequence. Similar probes are described in Mergny et al., Nucleic Acids Res., 22:920-928 (1994). Blok and Kramer, Molecular and Cellular Probes, 11: 187-194 (1997) describe an amplification RNA probe containing a molecular switch, i.e., a plurality of hairpin structures. Fujiwara and Oishi, Nucleic Acids Res., 26:5728-5733 (1998) describe a method of covalent attachment of probe DNA to double-stranded target DNA where an imperfect hairpin was used to hybridize to a target DNA. Sriprakash and Hartas, Gene Anal Techn., 6:29-32 (1989) describe a method of generating radioisotope labeled probe with hairpin nucleic acid structure. One common feature of the hairpin structure-containing probes described in the above references is that the nucleotide sequence complementary to a target nucleotide sequence always resides in the single-stranded, not double-stranded, segment of the hairpin structure.
The direct labeling approach can be problematic because nucleic acid labeling methods may fail to label different nucleic acids in a mixture equally. In addition, direct labeling may introduce mutations or other chemical modifications of the sample nucleic acid that prohibit or reduce hybridization.
Detection of hybridization in a microarray biochip by indirect labeling also can be problematic because background hybridization between the second probe may hybridize to the first probe immobilized on the microarray, giving rise to a high false-positive assay background. If the microarray utilizes only a single probe or very limited set of probes, the background may be reduced in the indirect labeling format by designing the specific second probe such that it does not hybridize to the immobilized probes on the array. However, when the microarray contains a wide variety of probe sequences for simultaneously detecting a variety of different nucleic acid targets (the reason for miniaturizing hybridization), designing second probes that are specific and that can avoid background hybridization to the immobilized probes becomes extremely difficult, if not impossible. Accordingly, a need exists for improved hybridization in general and for detecting hybridization on microarray formats in particular. The present invention addresses this and other related needs in the art.
In one aspect, the present invention provides an oligonucleotide probe for hybridization analysis, which probe comprises a nucleotide sequence which, under suitable conditions, is capable of forming a hairpin structure, wherein the double stranded segment of the hairpin structure is formed between two perfectly matched nucleotide sequences, and wherein at least a portion of the nucleotide sequence located within the double stranded segment is complementary to a target nucleotide sequence to be detected.
In another aspect, the present invention provides an array of oligonucleotide probes immobilized on a solid support for hybridization analysis, which array comprises a solid support suitable for use in nucleic acid hybridization having immobilized thereon a plurality of oligonucleotide probes, each of the probes comprising a nucleotide sequence which, under suitable conditions, is capable of forming a hairpin structure, wherein at least a portion of the nucleotide sequence located within the double stranded segment is complementary to a target nucleotide sequence to be detected.
In still another aspect, the present invention provides a method for detecting a target nucleotide sequence in a sample comprising the steps of: a) providing an oligonucleotide probe comprising a nucleotide sequence having a hairpin structure, with a double stranded segment, wherein the double stranded segment is formed between two complementary, preferably perfectly matched nucleotide sequences, and wherein at least a portion of the nucleotide sequence located within the double stranded segment is complementary to a target nucleotide sequence to be detected; b) contacting the probe provided in step a) with a sample containing or suspected of containing the target nucleotide sequence under conditions that favor intermolecular hybridization between the probe and the target nucleotide sequence over intramolecular hybridization of the probe itself; and c) assessing the intermolecular hybrid formed in step b), whereby presence of the intermolecular hybrid indicates the presence of the target nucleotide sequence in the sample.
In yet another aspect, the present invention provides a method for detecting a target nucleotide sequence in a sample comprising the steps of: a) providing an array of oligonucleotide probes immobilized on a solid support suitable for use in nucleic acid hybridization having immobilized thereon a plurality of oligonucleotide probes, each of the probes comprising a hairpin structure with a double stranded segment and wherein at least a portion of the nucleotide sequence located within the double stranded segment is complementary to a target nucleotide sequence to be detected; b) contacting the array of probes provided in step a) with a sample containing or suspected of containing the target nucleotide sequence under conditions that favor intermolecular hybridization between the probes and the target nucleotide sequence over intramolecular hybridization of the probes themselves; and c) assessing the intermolecular hybrids formed in step b), whereby presence of the intermolecular hybrids indicates the presence of the target nucleotide sequence in the sample.